Patients with CKD have increased vascular calcification and the pathogenesis parallels normal bone osteogenesis. We have previously demonstrated that uremia is a major risk factor and in vitro studies show that the effect of uremia on vascular smooth muscle cells (VSMC) de-differentiation is additive to that of phosphorus. Once the VSMC become osteoblast like, they secrete matrix vesicles (MV) that interact with the extracellular matrix (ECM). During our current VA Merit review, we have characterized the content of the MV and demonstrated that increased annexin-II and decreased fetuin-A content leads to increased ability to mineralize ECM. Furthermore, the formation of these MV requires an influx of calcium (extracellular to intracellular) and was dependent on signaling pathways involved in cytoskeleton structure and organization. Transglutaminase 2 (TG2) is a calcium dependent enzyme that is a co-receptor with ss1 integrins and fibronectin on the cell membrane, and therefore involved in the communication between the cytoskeleton and the ECM, can cross link ECM proteins, and can alter intracellular calcium. Our preliminary data demonstrates that TG2 activity is increased in VSMC from CKD compared to Normal rats, is highly expressed in calcifying areas of both human and rat arteries, and is expressed in MV. Our Hypothesis is: Increased transglutaminase (TG2) activity in CKD accelerates vascular calcification by inducing a pro-calcifying environment facilitating vascular smooth muscle cell-extracellular matrix interaction. In Aim 1, we will test the hypothesis that increased TG2 activity in CKD induces vascular smooth muscle cell de-differentiation with calcifying matrix vesicle (MV) formation and extracellular matrix cross-linking. We will determine the effect of blockade of TG2 activity in VSMC from CKD, compared to Normal animals on a) cellular differentiation to an osteoblast like phenotype, b) ECM protein synthesis and TG cross-linking of the ECM, c) cellular calcification and d) MV calcification potential and e) the interaction between VSMC/MV and the ECM. In Aim 2 we will test the hypothesis that increased extracellular calcium in CKD activates both membrane and cytosolic TG2 to enhance the interaction of VSMC/MV with the ECM by altering the composition of MV to a more pro-calcifying content. Finally, we will determine if inhibition of the increased TG2 activity in CKD can prevent arterial calcification in vivo. We will determine if oral administration of cystamine, a broad TG inhibitor, can inhibit aorta vascular calcification in a naturally occurring rat model of CKD-MBD (Chronic Kidney Disease-Mineral Bone Disorder). After initial short term dose finding studies, we will treat CKD rats with and without cystamine from 20 to 34 weeks and assess efficacy by determining calcification and TG2 activity of the thoracic aorta, and safety by determining bone calcium content and trabecular cross-linking by microCT. These studies will provide the needed pre-clinical in vitro and in vivo studies to determine if the administration of cystamine, a derivative of which is approved for other uses in humans, may halt progression of vascular calcification in CKD through its inhibition of transglutaminase activity.